MGMT and CALCA promoter methylation are associated with poor prognosis in testicular germ cell tumor patients.

Testicular germ cell tumors (TGCT) represent the second main cause of cancer-related death in young men. Despite high cure rates, refractory disease results in poor prognosis. Epigenetic reprogramming occurs during the development of seminomas and non-seminomas. Understanding the molecular and genetic basis of these tumors would represent an important advance in the search for new TGCT molecular markers. Hence the frequency of methylation of a gene panel (VGF, MGMT, ADAMTS1, CALCA, HOXA9, CDKN2B, CDO1 and NANOG) was evaluated in 72 primary TGCT by quantitative methylation specific PCR. A high frequency of MGMT (90.9%, 20/22; p=0.019) and CALCA (90.5%, 19/21; p<0.026) methylation was associated with non-seminomatous tumors while CALCA methylation was also associated with refractory disease (47.4%, 09/19; p=0.005). Moreover, promoter methylation of both genes predicts poor clinical outcome for TGCT patients (5-year EFS: 50.5% vs 77.1%; p=0.032 for MGMT and 51.3% vs 77.0%; p=0.029 for CALCA). The findings of this study indicate that methylation of MGMT and CALCA are frequent and could be used as new molecular markers of prognosis in TGCT.

Global and gene-specific DNA methylation pattern discriminates cholecystitis from gallbladder cancer patients in Chile.

AIM: The aim of the study was to evaluate the use of global and gene-specific DNA methylation changes as potential biomarkers for gallbladder cancer (GBC) in a cohort from Chile. MATERIAL & METHODS: DNA methylation was analyzed through an ELISA-based technique and quantitative methylation-specific PCR. RESULTS: Global DNA Methylation Index (p = 0.02) and promoter methylation of SSBP2 (p = 0.01) and ESR1 (p = 0.05) were significantly different in GBC when compared with cholecystitis. Receiver curve operator analysis revealed promoter methylation of APC, CDKN2A, ESR1, PGP9.5 and SSBP2, together with the Global DNA Methylation Index, had 71% sensitivity, 95% specificity, a 0.97 area under the curve and a positive predictive value of 90%. CONCLUSION: Global and gene-specific DNA methylation may be useful biomarkers for GBC clinical assessment.

Genome-wide methylation profiling reveals Zinc finger protein 516 (ZNF516) and FK-506-binding protein 6 (FKBP6) promoters frequently methylated in cervical neoplasia, associated with HPV status and ethnicity in a Chilean population.

Cervical cancer is a major health concern among women in Latin America due to its high incidence and mortality. Therefore, the discovery of molecular markers for cervical cancer screening and triage is imperative. The aim of this study was to use a genome wide DNA methylation approach to identify novel methylation biomarkers in cervical cancer. DNA from normal cervical mucosa and cervical cancer tissue samples from Chile was enriched with Methylated DNA Immunoprecipitation (MeDIP), hybridized to oligonucleotide methylation microarrays and analyzed with a stringent bioinformatics pipeline to identify differentially methylated regions (DMRs) as candidate biomarkers. Quantitative Methylation Specific PCR (qMSP) was used to study promoter methylation of candidate DMRs in clinical samples from two independent cohorts. HPV detection and genotyping were performed by Reverse Line Blot analysis. Bioinformatics analysis revealed GGTLA4, FKBP6, ZNF516, SAP130, and INTS1 to be differentially methylated in cancer and normal tissues in the Discovery cohort. In the Validation cohort FKBP6 promoter methylation had 73% sensitivity and 80% specificity (AUC = 0.80). ZNF516 promoter methylation was the best biomarker, with both sensitivity and specificity of 90% (AUC = 0.92), results subsequently corroborated in a Prevalence cohort. Together, ZNF516 and FKBP6 exhibited a sensitivity of 84% and specificity of 81%, when considering both cohorts. Our genome wide DNA methylation assessment approach (MeDIP-chip) successfully identified novel biomarkers that differentiate between cervical cancer and normal samples, after adjusting for age and HPV status. These biomarkers need to be further explored in case-control and prospective cohorts to validate them as cervical cancer biomarkers.

Association of promoter methylation of VGF and PGP9.5 with ovarian cancer progression.

PURPOSE: To elucidate the role of biological and clinical impact of aberrant promoter hypermethylation (PH) in ovarian cancer (OC). EXPERIMENTAL DESIGN: PH of PGP9.5, HIC1, AIM1, APC, PAK3, MGMT, KIF1A, CCNA1, ESR1, SSBP2, GSTP1, FKBP4 and VGF were assessed by quantitative methylation specific PCR (QMSP) in a training set. We selected two genes (VGF and PGP9.5) for further QMSP analysis in a larger independent validation (IV) set with available clinical data. Biologic relevance of VGF gene was also evaluated. RESULTS: PH frequency for PGP9.5 and VGF were 85% (316/372) and 43% (158/366) respectively in the IV set of samples while no PH was observed in controls. In 372 OC cases with available follow up, PGP9.5 and VGF PH were correlated with better patient survival [Hazard Ratios (HR) for overall survival (OS) were 0.59 (95% Confidence Intervals (CI)  = 0.42-0.84, p = 0.004), and 0.73 (95%CI = 0.55-0.97, p = 0.028) respectively, and for disease specific survival (DSS) were 0.57 (95%CI 0.39-0.82, p = 0.003) and 0.72 (95%CI 0.54-0.96, p = 0.027). In multivariate analysis, VGF PH remained an independent prognostic factor for OS (HR 0.61, 95%CI 0.43-0.86, p<0.005) and DSS (HR 0.58, 95%CI 0.41-0.83, p<0.003). Furthermore, PGP9.5 PH was significantly correlated with lower grade, early stage tumors, and with absence of residual disease. Forced expression of VGF in OC cell lines inhibited cell growth. CONCLUSIONS: Our results indicate that VGF and PGP9.5 PH are potential biomarkers for ovarian carcinoma. Confirmatory cohorts with longitudinal follow-up are required in future studies to define the clinical impact of VGF and PGP9.5 PH before clinical application.

Aberrant promoter methylation can be useful as a marker of recurrent disease in patients with cervical intraepithelial neoplasia grade III.

INTRODUCTION: Although studies of risk factor profiles have been conducted to identify biological markers to predict the natural history of cervical intraepithelial neoplasia (CIN) grade III, there is not sufficient information to support the routine clinical use of any biomarker. OBJECTIVES: The purpose of this study was to examine aberrant promoter methylation, which is implicated in cancer development and progression, in CIN III lesions in order to identify markers associated with more aggressive biological behavior that could be used to recognize women who are at higher risk of recurrence. PATIENTS AND METHODS: We used methylation-specific polymerase chain reaction to analyze promoter hypermethylation of 8 genes (p16, RARbeta, GSTP1, MGMT, p14, TIMP3, E-cad and DAPk) in 33 uterine cervix cones with CIN III that were also submitted to human papillomavirus (HPV) genotyping. All 33 patients in this study had been clinically followed after conization with Papanicolaou smears, colposcopy, and biopsy when indicated, every 6 months during 5 years. RESULTS: Of the 33 patients, 12 (36%) underwent immediate hysterectomy after conization for having compromised cone margins, 14 (43%) have not relapsed, and 7 (21%) presented CIN relapse. The frequency of HPV infection in this group was 97% and no significant difference between the groups was observed. HPV of high oncogenic risk was present in 29 (87.9%) cases; HPV 16 was the most frequent (69.7%), while HPV 18 was found in 33.3%; however, it was associated with HPV 16 in 15.1%. Concomitant infection by HPV 6/11 was detected in 21.2% (15.1% with HPV 16 and 6.1 with HPV 18). 85.7% (6/7) of patients with recurrence had HPV 18 vs 0% (0/14) of patients without recurrence (P = 0.0001). At least 1 of the 8 genes was found hypermethylated in all samples. Concomitant hypermethylation of several genes was frequently found. However, CIN relapse was only seen in the cases with hypermethylation of 3 or more of the 8 genes studied (P = 0.0039). CONCLUSION: We suggest that aberrant promoter methylation may play a role and may serve as a useful biomarker in the recurrence of CIN.

Aberrant methylation in pediatric myelodysplastic syndrome.

BACKGROUND: Aberrant methylation of gene promoter region is responsible for inappropriate gene silencing, and it has been associated to initiation and progression of cancer. Aberrant promoter methylation is frequently observed in adult patients with myelodysplastic syndrome (MDS), but in pediatric patients it has been poorly investigated. METHODS: We examined the promoter methylation status of 13 genes in bone marrow cells collected at diagnosis of 21 pediatric patients with MDS (subtype RAEB or RAEB-t). For this analysis, we performed sodium bisulfite treatment of genomic DNA, followed by methylation specific PCR (MSP). RESULTS: In pediatric MDS samples, we observed two genes frequently methylated: CALCA was methylated in 85.7% (18/21) of the analyzed samples and CDKN2B in 50% (6/12). CONCLUSIONS: Our findings indicate that CALCA and CDKN2B are frequently methylated in pediatric MDS. It suggests that aberrant methylation in pediatric MDS seems to be similar to adult MDS, thus pediatric patients could be also benefited with treatment using demethylating agents.

Differential expression of apoptosis related proteins and nitric oxide synthases in Epstein Barr associated gastric carcinomas.

AIM: To determine the incidence of Epstein Barr virus associated gastric carcinoma (GC) in Brazil and compare the expressions of apoptosis related proteins and nitric oxide synthases between EBV positive and negative gastric carcinoma. METHODS: In situ hybridization of EBV-encoded small RNA-1 (EBER-1) and PCR was performed to identify the presence of EBV in GCs. Immunohistochemistry was used to identify expressions of bcl-2, bcl-xl, bak, bax, p53, NOS-1, NOS-2, and NOS-3 proteins in 25 EBV positive GCs and in 103 EBV negative GCS. RESULTS: 12% of the cases of GC (25/208) showed EBER-1 and EBNA-1 expression. The cases were preferentially of diffuse type with intense lymphoid infiltrate in the stroma. EBV associated GCs showed higher expression of bcl-2 protein and lower expression of bak protein than in EBV negative GCs. Indeed, expressions of NOS-1 and NOS-3 were frequently observed in EBV associated GCs. CONCLUSION: Our data suggest that EBV infection may protect tumor cells from apoptosis, giving them the capacity for permanent cell cycling and proliferation. In addition, EBV positive GCs show high expression of constitutive NOS that could influence tumor progression and aggressiveness.

p16 gene methylation lacks correlation with angiogenesis and prognosis in multiple myeloma.

Methylation of p16 gene is a relatively frequent molecular finding in multiple myeloma (MM), but its clinical implication is disputable. Cell cycle regulators are now recognized as active in the control of angiogenesis, which is an integral component of pathogenesis and a target for new treatment modalities of this disease. On such background, we focused on determining whether loss of p16 function by methylation could be associated with increased angiogenesis and VEGF expression, and the prognostic relevance of p16 methylation in 50 untreated, newly diagnosed MM patients. Thirty-one percent (13/42) of 42 patients assessable for p16 gene methylation showed to be methylation-positive. High-angiogenesis was present in 73% of cases, but methylation of the p16 gene did not associate with angiogenesis or with VEGF expression. Also, p16 methylation did not show prognostic relevance or correlation with the clinical and laboratory parameters of prognostic significance in univariate analysis. P16 immunoexpression presented only a faint agreement with the molecular study. Therefore, p16 methylation seems to have no correlation with angiogenesis and VEGF expression, neither with overall and event-free survival in MM patients. Besides, P16 immunohistochemistry seems inadequate to substitute the molecular study of methylation in this type of tumor cells. Additional studies are needed to clarify the correspondence between the epigenetic alteration of the p16 gene and its protein immunexpression, and the clinical relevance of p16 methylation in MM patients.

Avaliação da detecção de hipermetilação de promotores gênicos como marcador molecular para o câncer de bexiga / Evaluation of the detection of hypermethylation of gene promoters as a molecular marker for bladder cancer

O câncer de bexiga é o quinto tipo de câncer mais incidente no mundo ocidental e as suas taxas de recidiva são altas. Os testes de detecção mais eficazes feitos atualmente são a cistoscopia (um método invasivo) e a citologia (apesar de não ser um método invasivo, a sua sensibilidade é baixa, principalmente em tumores superficiais). O silenciamento de genes devido à metilação de seus promotores parece ser uma via importante pela qual os tumores inativam os genes supressores de tumor. O desenvolvimento de marcadores moleculares para o câncer baseado na detecção de metilação anormal de promotores gênicos oferece uma abordagem promissora. Estudos recentes demonstraram que estas alterações podem ser detectadas tanto em amostras de tumores quanto em fluídos corpóreos. Para este estudo foram escolhidos 11 genes (DAPK, E-CAD, GSTP1, MGMT, p14, p16, RARß, TIMP-3, 14-3-3s, APC e H-CAD), que já haviam sido descritos como freqüentemente silenciados devido à metilação de seus promotores em outros tipos de tumores. O objetivo deste estudo foi investigar padrões de metilação destes genes em câncer de bexiga e correlacionar esses dados com parâmetros clínico-patológicos. Foi testada também a detecção de metilação em promotores gênicos em DNA extraídos de amostras de urina através de PCR específica para a detecção de metilação (MSP). Também buscou-se construir um modelo experimental para o estudo de hipermetilação por MSP em um promotor gênico nunca antes analisado. O padrão de metilação dos 11 genes foi determinada em 38 amostras de tumores de bexiga. Os DNAs foram extraídos de amostras emblocadas em parafina e submetidos à MSP. As freqüências de metilação encontradas nos tumores foram de: 68,4% para o gene 14-3-3s, 60,5% para o gene RARb, 57,9% para o gene APC, 42,1% para o gene E?CAD, 13,2% para o gene DAPK, 10,5% para os genes GSTP1, p14, p16 e H-CAD, 5,3% para o gene MGMT e 0% para o gene TIMP-3. A metilação do promotor do gene E?CAD foi correlacionada, com significância estatística, com a presença de metástases. A metilação do promotor do gene APC foi observada em tumores em estadios mais avançados. A metilação do promotor do gene 14-3-3s foi associada com o estágio mais avançado T1 quando comparado ao estágio menos avançado Ta. A detecção de metilação em promotores gênicos em DNAs extraídos de amostras de urina através de MSP foi factível. Foram desenhados primers para detectar o perfil de metilação do gene ADAM23 pela técnica de MSP e os resultados obtidos mostraram que esse evento não é freqüente na carcinogênese de bexiga. Os resultados do presente trabalho sugerem que o perfil de metilação de determinados genes pode ser usado como marcador molecular em potencial para o câncer de bexiga. No entanto, estudos mais amplos são necessários para a confirmação dos achados deste trabalho

Bladder cancer is the fifth most common cancer in the westem world and its re,turrence rates are high. The most used methods to detect and monitOr bladder cancer are cystoscopy (an invasive procedure) and urine cytoiogy (although it is a noninvasive method, its sensibility is low, especially for superficial tumors). Methylation-associated silencing of genes is considered as an important pathway to inactivate tumor supplessor genes in cancer. The development of tumor markers based on the detection of aberrant promoter methylation offers a potentially powerfirl approach for the detection of this disease. Recent studies demonstrated that these alterations could be detected not only in tumors, but also in bodily fluids. We selected 11 genes (DAPK, E-CAD, GSTPl, MGMT, pl'\, p16, MRf'TIMP-3' l1-3-3o, APC e H-CAD), frequently silenced by abenant methylation in many different tumor types, and investigated their aberrant methylation profile in bladder cancers. We tried to correlate our findings with clinicopathological parameters. We also investigated the feasibility of the detection of neoplastic cells in the urine using methylation analysys. We also aimed to determine an experimental model to detect the methylation status of a gene, which had never been analysed by the methylation specific PcR (MSP) before. The methylation status of the 11 selected genes was determined in 38 bladder cancer samples' DNA was extracted from formaline-fixed, paraffin-embedded sections and the DNA was stLbjected to MSP. Methylation frequencies of the tested genes wele 68,4%o for 11-i-3o, 6l),5% for MRf , 57,9o/o for APC, 42,1o/o for E-CAD, 13,2% for DAPK, 10,5% for GSTPl, p.t1, p\6 and H-CAD,5,3o/o MGMT for and \Vo for TIMP-3. E-CAD promoter methylation was significantly conelated with the prcsence of metastasis. IPC promoter methylation was olrserved in tumors in higher stages. l1-i-3d promoter methylation was associated with the hr gher stage T1 when compared to the lower stage Ta. We were also able to detect the promoter methylation profile ofthe selected genes by analyzing urine samples. We designed pr:imers to detect the promoter methylation status of the ADAM23 gene by the MSP techlique and these results showed that this event is not frequent in bladder cancer. The rosults presented here suggest that the methylation profile may be useful as a potencial b ladder cancer biomarker. Further studies are needed to confirm our findings'